An understanding of the mechanisms involved in the regulation of tyrosine hydroxylase (TH:EC 1.14.16.2), the enzyme which catalyzes the rate-limiting step in the biosynthesis of the catecholamines, may lead to insights into the cellular events that occur, or are prevented from occurring, during certain physiological states and disorders, such as depression, schizophrenia, hypertension and stress. Presented in this proposal are a series of studies designed to investigate the cyclic AMP-mediated and glucocorticoid-mediated mechanisms responsible for the induction of TH in mouse neuroblastoma and rat pheochromocytoma cell lines. The specific aims of this proposal are to study the following aspects of the induction of TH: (1) the effects of these inducing agents on the processes that determine the intracellular level of TH; these processes include the rates of synthesis and degradation of TH, the functional activity and cellular levels of mRNA for TH (mRNATH), and the processes that influence these mRNATH parameters; and (2) the mechanism(s) by which cyclic AMP acts to enhance the rate of transcription of the gene for TH. To accomplish these aims, we have produced antiserum to TH and a cDNA probe complementary to the mRNATH. With these tools we can measure the effects of cyclic AMP and glucocorticoids on the processes described above. The cyclic AMP-mediated incorporation of 32P-phosphate into specific nuclear proteins, fractionated by gel electrophoresis techniques, will be correlated with the cyclic AMP-mediated increase in the rate of transcription of the gene for TH. The protein kinase(s) or protein phosphatase(s) responsible for the phosphorylation of the nuclear proteins implicated in the induction of mRNATH will be identified by comparing the nuclear protein(s) phosphorylated by isolated protein kinases or dephosphorylated by isolated protein phosphatases in cell-free experiments using nuclei isolated from the cells. The possible involvement of the regulatory subunits of cyclic AMP-dependent protein kinase or other cyclic AMP-binding proteins in the activation of the TH gene will also be investigated, if the phosphorylation of a nuclear protein does not correlate with the induction of mRNATH. The proposed studies will increase our understanding of the biochemical events responsible for the induction of TH and may be helpful in elucidating the mechanisms responsible for the transsynaptic induction of TH that occurs in vivo following prolonged stress.